Methods and reagents for detection of bacteria in cerebrospinal fluid

ABSTRACT

PCT No. PCT/US92/06365 Sec. 371 Date May 16, 1994 Sec. 102(e) Date May 16, 1994 PCT Filed Jul. 31, 1992 PCT Pub. No. WO93/03186 PCT Pub. Date Feb. 18, 1993Methods and reagents are provided for detecting bacterial nucleic acids in cerebrospinal fluid. In a preferred embodiment, a panel of probes is provided for detecting and identifying causal agents of meningitis.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 07/738,393,filed Jul. 31, 1991 now abandoned, which is a continuation-in-part ofapplications U.S. Ser. No. 07/593,176, filed Oct. 5, 1990, nowabandoned, and U.S. Ser. No. 07/696,448, filed May 6, 1991 nowabandoned.

TECHNICAL FIELD

The present invention relates generally to methods and reagents foridentifying and detecting bacteria in cerebrospinal fluid (CSF).

BACKGROUND OF THE INVENTION

In order to treat successfully a disease caused by a bacterium, therapid and accurate detection and identification of the disease-causingbacterium is required. The detection and identification havetraditionally been accomplished by pure culture isolation andidentification procedures that make use of knowledge of specimen source,growth requirements, visible (colony) growth features, microscopicmorphology, staining reactions, and biochemical characteristics.

A number of different bacterial species can cause meningitis whenpresent in the CSF. The species most frequently causing meningitisinclude: Escherichia coli and other enteric bacteria, Haemophilusinfluenzae, Neisseria meningitidis, Streptococcus pneumoniae,Streptococcus agalactiae, and Listeria monocytogenes.

Conventional methods of detection and identification of bacteria incerebrospinal fluid include the Gram stain, latex agglutination andother antibody-based tests, and culture. The Gram stain andantibody-based tests are rapid (<1 hour), but of low sensitivity(requiring at least 10⁴ colony forming units [CFU]bacteria per ml).Culture methods, while sensitive to approximately 2 CFU per ml, requireovernight incubation.

A number of scientific publications relating to this invention exist.For example, the polymerase chain reaction has been used to detectindividual species of bacteria causing meningitis: Kuritza and Oehler,May, 1991, Abstracts of the General Meeting of the ASM page 84; Deneerand Boychuk, 1991, Applied Environmental Microbiology 57: 606-609; andKristiansen et al., 1991, Lancet 337: 1568-1569.

In addition, some of the nucleotide sequence data used herein isavailable in Genbank. The method of reverse dot-blot detection has beendescribed by Saiki et al., 1989. The use of uracil-N-glycosylase hasbeen described by Longo et al., 1990, Gene 93: 125-128.

A method of detecting bacteria in Cerebrospinal Fluid ("CSF") which isboth sensitive and rapid would represent a great improvement overcurrent methods of detection. The present invention meets these needs.

SUMMARY OF THE INVENTION

The present invention pertains to methods and reagents for the rapiddetection and identification of bacteria in CSF. The detection andidentification is based upon the hybridization of nucleotide probes tonucleotide sequences present in a defined species or group of species,but not in others.

In a preferred method, a target region from genomic DNA or complementaryDNA transcribed from 16S rRNA is amplified and the resultant amplifiedDNA is treated with a panel of probes. Each probe in the panel canhybridize to the DNA of a different species or group of species ofbacteria found in CSF. The probe which successfully hybridizes to theamplified DNA is determined and the bacterium is classified as aparticular species or group of species.

The invention also pertains to specific probes and their complements foridentifying bacteria found in CSF. It also pertains to uniqueoligonucleotide sequences, and mutants, fragments and subsequencesthereof, from which such specific probes were derived.

As indicated, also contemplated herein is a panel of probes which willallow the detection and identification of bacteria commonly found inCSF. The panel includes probes for the bacteria causing meningitislisted above as well as bacterial species which are commonly consideredcontaminants of human clinical samples such as blood or cerebrospinalfluid. Such contaminant species are also capable of causing meningitis;however, these organisms do so at a lower frequency than the agentslisted in the "Background of the Invention" and include: Bacillusspecies, Corynebacterium species, Propionibacterium acnes and otherPropionibacterium species, and Staphylococcus epidermidis and othercoagulase-negative Staphylococci (Bergey's Manual of SystematicBacteriology, ed. J. G. Holt, Williams and Wilkins, Baltimore, Md.,which is incorporated herein by reference).

The panel will also include a probe for a wide range of bacteria,referred to as a "universal bacterial probe," in order to detect speciesthat cause meningitis at a lower frequency but for which there is nospecific probe included on the panel. Therefore, this probe will provideconfirmation of the detection of pathogens and contaminants as well asdetection of species for which there is no specific probe.

Further defined herein are nucleotide sequence data for some of thepathogen and contaminant species in the region of the 16S rRNA. Suchnucleotide sequence information for the 16S rRNA gene is not availablefor some of the above bacterial species. Accordingly, it was necessaryto obtain the sequence information experimentally.

The invention further provides methods of amplification and associatedreagents for kits containing universal bacterial primers for amplifyinga specific universal target region of DNA for all bacteria and probeswhich hybridize to a nucleotide sequence which is characteristic of aspecies or group of species of bacteria within that target region.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows nucleotide sequence data for part of the 16S rRNA gene for:

1-1) Neisseria meningitidis

1-2) Streptococcus agalactiae

1-3) Streptococcus pneumoniae

1-4) Staphylococcus epidermidis

1-5) Staphylococcus aureus

1-6) Streptococcus pyogenes

FIG. 2 shows nucleotide sequences and positions of oligonucleotideprobes for Streptococcus agalactiae, Listeria monocytogenes,Streptococcus pneumoniae, Haemophilus influenzae, Neisseriamemingitidis, Escherichia coil, Propionibacterium acnes, Staphylococcusepidermidis, Staphylococcus aureus, Propionibacterium species, Bacillusspecies, coagulase-negative Staphylococci, and Corynebacterium species.

FIG. 3 shows a summary of data obtained from probe testing againstvarious bacterial DNAs as described in Example 2.

FIG. 4 shows a summary of data obtained from probe testing againstvarious bacterial DNAs as described in Example 4.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is a method for determining the presence of andidentifying bacteria by means of hybridizing probes to amplifiednucleotide sequences which are characteristic of a species or group ofspecies of bacteria.

The use of specific polynucleotide sequences as probes for therecognition of infectious agents is becoming a valuable alternative toproblematic immunological identification assays. For example, PCTpublication WO84/02721, published 19 July 1984 describes the use ofnucleic acid probes complementary to targeted nucleic acid sequencescomposed of ribosomal RNA, transfer RNA, or other RNA in hybridizationprocedures to detect the target nucleic acid sequence. While this assaymay provide greater sensitivity and specificity than known DNAhybridization assays, hybridization procedures which require the use ofa complementary probe are generally dependent upon the cultivationand/or enrichment of a test organism and are, therefore, unsuitable forrapid diagnosis. Probes can be used directly on clinical specimens if ameans of amplifying the DNA or RNA target is available.

Polymerase chain reaction (PCR) is a powerful nucleic amplificationtechnique that can be used for the detection of small numbers ofpathogens whose in vitro cultivation is difficult or lengthy, or as asubstitute for other methods which require the presence of livingspecimens for detection. In its simplest form, PCR is an in vitro methodfor the enzymatic synthesis of specific DNA sequences, using twooligonucleotide primers that hybridize to opposite strands and flank theregion of interest in the target DNA. A repetitive series of cyclesinvolving template denaturation, primer annealing, and the extension ofthe annealed primers by DNA polymerase results in the exponentialaccumulation of a specific fragment whose termini are defined by the 5'ends of the primers. PCR reportedly is capable of producing a selectiveenrichment of a specific DNA sequence by a factor of 10¹². The PCRmethod is described in Saiki et al., 1985, Science 230: 1350, and is thesubject of U.S. Pat. Nos. 4,683,195, 4,683,202, 4,800,159, and4,965,188, which are incorporated herein by reference. This method hasbeen used to detect the presence of the aberrant sequence in thebeta-globin gene which is related to sickle cell anemia (Saiki et al.,1985, supra.) and human immunodeficiency virus (HIV) RNA (Byrne et al.,1988, Nuc. Acids Res. 16: 4165, and U.S. Pat. No. 5,008,182,incorporated herein by reference).

The invention provides methods for determining the presence of abacterial polynucleotide in a sample suspected of containing saidpolynucleotide, wherein said polynucleotide contains a selected targetregion, said method comprising: (a) amplifying the target region, ifany, to a detectable level; (b) providing a panel of polynucleotideprobes, each containing a sequence which is complementary to apolynucleotide sequence characteristic of a different species or groupof species of bacteria in the target region: (c) incubating theamplified target region, if any, with the polynucleotide probes underconditions which allow specificity of hybrid duplexes: and (d) detectinghybrids formed between the amplified target region, if any, and thepolynucleotide probes.

The methods of the present invention thus enable determination of thepresence of the bacteria more rapidly than heretofore possible withprior art detection methods. The basic PCR process is carried out asfollows.

A sample is provided which needs to be tested or is suspected ofcontaining a particular nucleic acid sequence of interest, the "targetsequence." The nucleic acid contained in the sample may be first reversetranscribed into cDNA, if necessary, and then denatured, using anysuitable denaturing method, including physical, chemical, or enzymaticmeans, which are known to those of skill in the art. A preferredphysical means for strand separation involves heating the nucleic aciduntil it is completely (>99%) denatured. Typical heat denaturationinvolves temperatures ranging from about 80° C. to about 150° C., fortimes ranging from about 5 seconds to 10 minutes using currenttechnology. Methods for the amplification of RNA targets using athermostable DNA polymerase are described in PCT/US90/07641, filed Dec.21, 1990, and incorporated herein by reference.

The denatured DNA strands are then incubated with the selectedoligonucleotide primers under hybridization conditions, conditions whichenable each primer to hybridize to a single-stranded nucleic acidtemplate. As known in the art, the primers are selected so that theirrelative positions along a duplex sequence are such that an extensionproduct synthesized from one primer, when it is separated from itscomplement, serves as a template for the extension of the other primer.

The primer must be sufficiently long to prime the synthesis of extensionproducts in the presence of the agent for polymerization. The exactlength of the primers will depend on many factors, includingtemperature, source of the primer and use of the method. For example,depending on the complexity of the target sequence, the oligonucleotideprimer typically contains about 15-30 nucleotides, although it maycontain more or fewer nucleotides. Short primer molecules generallyrequire cooler temperatures to form sufficiently stable hybrid complexeswith the template. The primers must be sufficiently complementary tohybridize selectively with their respective strands.

The primers used herein are selected to be "substantially" complementaryto the different strands of each specific sequence to be amplified. Theprimers need not reflect the exact sequence of the template, but must besufficiently complementary to hybridize selectively with theirrespective strands. Non-complementary bases or longer sequences can beinterspersed into the primer, provided that the primer retainssufficient complementarity with the sequence of one of the strands to beamplified to hybridize therewith, and to thereby form a duplex structurewhich can be extended by the agent for polymerization. Thenon-complementary nucleotide sequences of the primers may includerestriction enzyme sites. Appending a restriction enzyme site to theend(s) of the target sequence is particularly helpful for subsequentcloning of the target sequence.

The oligonucleotide primers and probes may be prepared by any suitablemethod. For example, synthetic oligonucleotides can be prepared usingthe triester method of Matteucci et al., 1981, J. Am. Chem. Soc. 03:3185-3191. Alternatively automated synthesis may be preferred, forexample, on a Biosearch 8700 DNA synthesizer using cyanoethylphosphoramidite chemistry. Many methods for labelling nucleic acids,whether probe or target, are known in the art and are suitable forpurposes of the present invention. Suitable labels may provide signalsdetectable by fluorescence, radioactivity, colorimetry, X-raydiffraction or absorption, magnetism, enzymatic activity, and the like.Suitable labels include fluorophores, chromophores, radioactive isotopes(particularly ³² P and ¹²⁵ I), electrondense reagents, enzymes andligands having specific binding partners. Enzymes are typically detectedby their activity. For example, horse-radish-peroxidase (HRP) can bedetected by its ability to convert diaminobenzidine to a blue pigment. Apreferred method for HRP based detection uses tetramethyl-benzidine(TMB) as described in Clin. Chem. 833: 1368 (1987). An alternativedetection system is the Enhanced Chemiluminescent (ECL) detection kitcommercially available from Amersham. The kit is used in accordance withthe manufacturer's directions.

Primers and probes are typically labeled with radioactive phosphorous ³²P by treating the oligonucleotides with polynucleotide kinase in thepresence of radiolabeled ATP. However, for commercial purposesnon-radioactive labeling systems may be preferred, such as, horseradishperoxidase-avidin-biotin or alkaline phosphatase detection systems. Ifthe primer or one or more of the dNTPs utilized in a PCR amplificationhas been labeled (for instance, the biotinylated dUTP derivativesdescribed by Lo et al., 1988, Nuc. Acids Res. 16: 8719) instead of theprobe, then hybridization can be detected by assay for the presence oflabeled PCR product. Biotinylated primers can be prepared by directbiotinylation of the oligonucleotide. For 5' biotinylation ofoligonucleotides during direct solid phase synthesis biotin-containingphosphoramidites were used according to Alves et al., 1989, Tetra. Let30: 3098: Cocuzza, 1989, Tetra Let. 30: 6287; and Barabino et al., 1989,EMBO J. 8: 4171. Solid phase synthesis of biotinylated oligonucleotidesat any internal or terminal (5' or 3') position is also suitable forpreparing biotinylated primers and probes (Pieles et al., 1989, NAR 18:4355, and Misiura et al., 1989, NAR 18: 4345). Alternatively, primerscan be biotinylated using terminal deoxynucleotide transferase (TdT)(Boeringer Mannheim).

Template-dependent extension of the oligonucleotide primer(s) iscatalyzed by a polymerizing agent in the presence of adequate amounts ofthe four deoxyribonucleoside triphosphates (dATP, dGTP, dCTP, and dTTP)or analogs such as dUTP, in a reaction medium which is comprised of theappropriate salts, metal cations, and pH buffering system. Suitablepolymerizing agents are enzymes known to catalyze primer- andtemplate-dependent DNA synthesis. Known DNA polymerases include, forexample, E. coli DNA polymerase I or its Klenow fragment, T₄ DNApolymerase, Taq DNA polymerase, DNA polymerase from Pyrococcus furiosus,Thermus thermophilus (Tth), Thermotoga maritima, Thermosipho africanus,and DNA polymerase from Thermococcus litoralis. The reaction conditionsfor catalyzing DNA synthesis with these DNA polymerases are well knownin the art.

The products of the synthesis are duplex molecules consisting of thetemplate strands and the primer extension strands, which include thetarget sequence. These products, in turn, serve as templates for anotherround of primer extension. In the second cycle, the primer extensionstrand of the first cycle is annealed with its complementary primer;synthesis yields a "short" product which is bounded on both the 5'-andthe 3'-ends by primer sequences of their complements. Repeated cycles ofdenaturation, primer annealing, and extension result in the exponentialaccumulation of the target region defined by the primers. Sufficientcycles are run to achieve the desired amount of polynucleotidecontaining the target region of nucleic acid. The desired amount mayvary, and is determined by the function which the product polynucleotideis to serve.

The PCR method can be performed in a number of temporal sequences. Forexample, it can be performed step-wise, where after each step newreagents are added, or in a fashion where all of the reagents are addedsimultaneously, or in a partial step-wise fashion, where fresh reagentsare added after a given number of steps.

In a preferred method, the PCR reaction is carried out as an automatedprocess which utilizes a thermostable enzyme. In this process thereaction mixture is cycled through a denaturing step, a primer annealingstep, and a synthesis step. A DNA thermal cycler specifically adaptedfor use with a thermostable enzyme may be employed, which utilizestemperature cycling without a liquid-handling system, therebyeliminating the need to add the enzyme at every cycle. This type ofmachine is commercially available from Perkin Elmer (Norwalk, Conn.).

After amplification by PCR, the target polynucleotides may be detecteddirectly by gel analysis provided the target DNA is efficientlyamplified and the primers are highly specific to the target region to beamplified. To assure PCR efficiency, glycerol and other related solventssuch as dimethyl sulfoxide, can be used to increase the sensitivity ofthe PCR at the amplification level and to overcome problems pertainingto regions of DNA having strong secondary structure. These problems mayinclude (1) low efficiency of the PCR, due to a high frequency oftemplates that are not fully extended by the polymerizing agent or (2)incomplete denaturation of the duplex DNA at high temperature, due tohigh GC content. The use of such solvents can increase the sensitivityof the assay at the level of amplification to approximately severalfemtograms of DNA (which is believed to correspond to a single bacterialcell). This level of sensitivity eliminates the need to detect amplifiedtarget DNA using a probe, and thereby dispenses with the requirementsfor labeling of probes, gel electrophoresis, Southern blotting, filterhybridization, washing and autoradiography. The concentration range forglycerol is about 5%-20% (v/v), and the DMSO concentration range isabout 3%-10% (v/v).

Alternatively, the target polynucleotides may be detected byhybridization with a polynucleotide probe which forms a stable hybridwith that of the target sequence under high to low stringencyhybridization and wash conditions. An advantage of detection byhybridization is that, depending on the probes used, additionalspecificity is possible. If it is expected that the probes will becompletely complementary (i.e., about 99% or greater) to the targetsequence, high stringency conditions will be used. If some mismatchingis expected, for example if variant strains are expected with the resultthat the probe will not be completely complementary, the stringency ofhybridization may be lessened. However, conditions are chosen which ruleout nonspecific/adventitious binding. Conditions which affecthybridization and which select against nonspecific binding are known inthe art (Molecular Cloning A Laboratory Manual, second edition, J.Sambrook, E. Fritsch, T. Maniatis, Cold Spring Harbor Laboratory Press,1989) Generally, lower salt concentration and higher temperatureincrease the stringency of binding. For example, in general, stringenthybridization conditions include incubation in solutions which containapproximately 0.1×SSC, 0.1% SDS, at about 65° C. incubation/washtemperature, and moderately stringent conditions are incubation insolutions which contain approximately 1-2×SSC, 0.1% SDS and about 50°C.-65° C. incubation/wash temperature. Low stringency conditions are2×SSC and about 30° C.-50° C.

Stringency requirements can be modified to alter target specificity asdescribed. For example, where Staphylococcus aureus is to be detected,it is well within the scope of the invention for those of ordinary skillin the art to modify the stringency conditions described above and causeother Staphylococcus species to be excluded or included as targets. Thenovel 16S rRNA sequences provided herein are suitable for preparing avast number of probe compounds having particular hybridizationcharacteristics as desired.

An alternate method of hybridization and washing is to perform a lowstringency hybridization (5×SSPE, 0.5% SDS) followed by a highstringency wash in the presence of 3M tetramethyl-ammonium chloride(TMACl). The effect of the TMACl is to equalize the relative binding ofA-T and G-C base pairs so that the efficiency of hybridization at agiven temperature corresponds more closely to the length of thepolynucleotide. Using TMACl, it is possible to vary the temperature ofthe wash to achieve the level of stringency desired. (See Basecomposition-independent hybridization in tetramethylammonium chloride: Amethod for oligonucleotide screening of highly complex gene libraries(Wood et al., 1985, Proc. Natl. Acad. Sci. USA 82: 1585-1588,incorporated by reference herein).

Probes for bacterial target sequences may be derived from the 16S rRNAgene sequences or their complements. The probes may consist of the basesA, G, C or T or analogs (including inosine and 5-methyl-cytosine). Theprobes may be of any suitable length which spans the target region andwhich allows specific hybridization to the target region. As used herein"specific hybridization" refers to that hybridization pattern orcharacter suitable for accurately identifying bacterial agents presentin a sample. In a preferred embodiment, the invention is suitable foruse as a panel array of probes. Consequently, the specific hybridizationpattern for the panel is a composite of individual specifichybridization probes, which probes may individually include or excludeparticular species, subtypes, or genera as desired. Thus, it may bepreferable to prepare probes for specifically identifying each ofStreptococcus agalactiae and S. pneumoniae. Alternatively, it may besuitable to prepare one probe for detecting any Streptococcus species.

Generally, the probes will have at least 14 nucleotides, preferably atleast 18 nucleotides, and more preferably at least 20 to 30 nucleotidesof either of the complementary DNA target strands. If there is to becomplete complementarity, i.e., if the strain contains a sequenceidentical to that of the probe, the duplex will be relatively stableunder even stringent conditions and the probes may be short, i.e., inthe range of about 10-30 base pairs. If some degree of mismatch isexpected with the probe, i.e., if it is suspected that the probe willhybridize to a variant region, or to a group of sequences such as allspecies within a specific genus, e.g., Bacillus species, the probe maybe of greater length (i.e., 15-40 bases) to balance the effect of themismatch(es).

The probe may be formed from a subset of the target region and thereforeneed not span the entire target region. Any subset of the target regionhas the potential to specifically identify the target region.Consequently, the nucleic acid probe may be 10-40 nucleotides in lengthand hybridize to as few as 10 nucleotides of the target region. Further,fragments of the probes may be used so long as they are sufficientlycharacteristic of the bacterial species to be detected. If desired, theprobe may also be labeled. A variety of labels which would beappropriate, as well as methods for their inclusion in the probe areknown in the art and include, for example, radioactive atoms, such as ³²P, or other recognizable functionalities, e.g., biotin (preferably usinga spacer arm), fluorescent dyes, electrondense reagents, enzymes capableof forming easily detectable reaction products (e.g., alkalinephosphatase and horseradish peroxidase), or antigens for which specificantisera or monoclonal antibodies are available. The probe may also bemodified for use in a specific format such as the addition of 100-150 Tresidues for reverse dot blot or the conjugation to Bovine serum albuminfor the method of Longiaru et al. described below.

It may be desirable to determine the length of the PCR product detectedby the probe. This may be particularly true if it is suspected thatvariant bacterial strains may contain deletions or insertions within thetarget region, or if one wishes to confirm the length of the PCR productIn such circumstances, it is preferable to subject the products to sizeanalysis as well as hybridization with the probe. Methods fordetermining the size of nucleic acids are known in the art, and include,for example, gel electrophoresis, sedimentation in gradients, and gelexclusion chromatography.

In order to obtain probes to be used for the PCR assays describedherein, enough of the nucleotide sequence of the target region must beknown. Analysis of the nucleotide sequence of the target region may beby direct analysis of the PCR amplified products as described inGyllensten and Erlich, 1988, Proc. Natl. Acad. Sci. USA 85: 7652, and inU.S. Pat. Nos. 5,065,584 and 5,075,216, which are incorporated herein byreference. A modification of this procedure involves separating theduplex DNA strands of the target region and generating a single strandedDNA template for use in the sequencing reactions (Mitchell and Merrill,1989, Analytical Biochemistry 178: 239-242).

One embodiment of the present invention is the discovery Of thenucleotide sequence data for the target regions (16S rRNA genes)obtained experimentally for the following organisms: Neisseriameningitidis (SEQ ID No. 28) Streptococcus pneumoniae (SEQ ID No. 30)and Streptococcus agalactiae (SEQ ID No. 29) and Staphylococcusepidermidis (SEQ ID No. 31), Streptococcus pyogenes (SEQ ID No. 48), andStaphylococcus aureus (SEQ ID No. 47). In FIG. 1--1 through 1--6 an "R"indicates A or G; "Y" indicates C or T; and "N" indicates A, G, T, or C.This information unexpectedly provided the nucleotide sequencevariability on which the species-specific or group-specific probes werebased. Fragments, subsequences, complements, or transcripts of thesenucleotide sequences will also be useful for design of these probes. Forexample, methods for preparing probes for hybridizing to detect thenovel 16S RNA nucleic acid sequences provided are disclosed herein.Suitable subsequence lengths for target-specific detection are between14 and 400 nucleotides. The novel sequences are also suitable forpreparing primers for amplification of the 16S rRNA target nucleicacids.

Diagnosis of bacterial meningitis using PCR is possible using a numberof different strategies. The following features of bacterial meningitisare relevant to the design of a diagnostic assay: (1) since CSF is anormally sterile body fluid, any level of infection by any species ofbacteria can result in meningitis; (2) since, in a given CSF sample froma patient with meningitis, one or more of a number of differentbacterial species may be present, any one species representing aclinical problem, it is important to be able to detect the presence ofmore than one bacterial species in the sample; and (3) the optimalantibiotic treatment varies depending on the type of bacterial speciescausing the meningitis. Hence, it is clinically useful to be able todifferentiate the individual species or groups of species capable ofcausing meningitis.

One approach to diagnosing bacterial meningitis is to run severaldifferent individual PCR assays. PCR detection of individual species ofbacteria causing meningitis has been described in the scientificliterature. For example, Kuritza and Oehler, May, 1991, Abstracts of theGeneral Meeting of the ASM page 84; Deneer and Boychuk, 1991, Appliedand Environmental Microbiology 57: 606-609; and Kristiansen, 1991,Lancet 337: 1568-1569.

Another approach, which is a preferred embodiment of the presentinvention, is to run a single PCR assay utilizing universal bacterialPCR primers and a panel of probes. Each probe is specific to a speciesor group of species which are commonly found in CSF and will preferablybe used simultaneously with other probes. The universal bacterialprimers correspond to highly conserved regions of a gene found in mostbacteria and hence are capable of amplifying the target gene of mostbacterial species. In a preferred embodiment, the primers used are thosedescribed in co-pending application, Ser. No. 07/696,448 incorporatedherein by reference. These primers, RW01 (SEQ ID No. 27)5'-AACTGGAGGAAGGTGGGGAT-3' and DG74 (SEQ ID No. 26)5'-AGGAGGTGATCCAACCGCA-3', yield an approximately 370 base pair PCRproduct corresponding to base pairs 1170 to 1540 of the E. coli 16S rRNAgene. This target region is of sufficient length to encompass tworegions of high variability characterized for the 16S rRNA gene,variable regions 8 and 9. The variability in these regions may encompassprobes which are to some degree specific to the various species andgroups of species of bacteria found in CSF.

The degree of specificity desired for each probe is dictated by twomajor considerations (1) the probe should be broad enough in range todetect most of the strains of a given species which are found inclinical samples and (2) the probe should be narrow enough in range toexclude closely related species that are commonly found in cerebrospinalfluid. In some cases, a probe that is broad in range and detects someclosely related species that are not found in cerebrospinal fluid ispreferable to a narrower range probe that may not detect all the strainsdesired. Information on (1) the types of bacterial species found incerebrospinal fluid and (2) species closely related to a given speciesare described in Bergey's Manual of Systematic Bacteriology (ed. J. G.Holt, Williams and Wilkins, Baltimore, Md.) and The Manual of ClinicalMicrobiology (ed. A. Balows, American Society for Microbiology,Washington, D.C.).

For the probes described, the prior art, while providing guidelines forthe characteristics of optimal probes (such as the sequence incomparison to available nucleotide sequence data, a low degree ofsecondary structure and optimal length) does not provide a means ofpredicting the experimental performance of probes for detecting bacteriafound in CSF. This information must be discovered empirically byhybridization testing of many different isolates of the pathogens and ofclosely related species, as illustrated in the examples below. Thenucleotide sequences described in FIG. 2 provide preferred embodimentsof the invention. However, providing the specific sequences and methodsshown herein, one of ordinary skill in the art is enabled to prepareadditional probes that are within the scope of the present invention.

In addition to probes which allow species- or group-specificidentification of bacteria, the panel of probes would also preferablyinclude a universal bacterial probe capable of specifically hybridizingto the amplified target region of any bacterial species (RDR245,5'-GTACAAGGCCCGGGAACGTATTCACCG-3' [SEQ ID No. 37], described incopending application Ser. No. 07/696,448, filed May 6, 1991 nowabandoned, incorporated herein by reference). This universal bacterialprobe detects the presence of bacteria not detected by the more specificprobes of the invention such as species representing less common causesof meningitis, for example, Flavobacterium meningosepticum, etc. Thepanel of probes preferably includes probes of bacterial species commonlyconsidered contaminants of clinical samples, such as Corynebacteriumspecies, Bacillus species, Propionibacterium species, andcoagulase-negative Staphylococci. The panel could also include otherprobes which are relatively broad in their range of detection, such asRW03 (SEQ ID No. 43), for gram-positive bacteria, and RDR476 (SEQ ID No.44) and RDR477 (SEQ ID No. 45), which used together detect gram-negativebacteria other than Flauobacteria and Bacteroides.

RW03 SEQ ID No. 43 5'-GACGTCAAATCATCATGCCCCTTATGTC-3'

RDR476 SEQ ID No. 44 5'-GACCTAAGGGCCATGATGACTYGACGTC-3'

RDR477 SEQ ID No. 45 5'-GACATAAGGGCCATGAGGACTTGACGTC-3'

The presence of the target sequence in a biological sample is detectedby determining whether a hybrid has been formed between the probe andthe nucleic acid subjected to the PCR amplification techniques. Methodsto detect hybrids formed between a probe and a nucleic acid sequence areknown in the art. For example, an unlabeled sample may be transferred toa solid matrix to which it binds, and the bound sample subjected toconditions which allow specific hybridization with a labeled probe: thesolid matrix is then examined for the presence of the labeled probe. Inthe disclosed embodiments of the invention where the hybridizationtarget nucleic acids, i.e., PCR product DNA, is fixed to a solidsupport, the term Format I may be used to describe such a detectionscheme.

Alternatively, if the sample is labeled, an unlabeled probe is bound tothe matrix, and after exposure to the labeled sample under theappropriate hybridization conditions, the matrix is examined for thepresence of a label. Saiki et at., 1989, Proc. Natl. Acad. Sci. USA 86:6230-6234, which is incorporated herein by reference, describe methodsof immobilizing multiple probes on a solid support and usinghybridization to detect the amplified target polynucleotides of interest(see also copending U.S. Ser. No. 07/414,542, filed Sep. 29, 1989, U.S.Pat. No. 5,232,829 which is incorporated herein by reference).

The latter two procedures are well suited to the use of a panel ofprobes which can provide simultaneous identification of more than onepathogen or contaminant in a single clinical sample. As used herein,"Format II" refers to a detection scheme wherein the oligonucleotideprobe is fixed to a solid support. In another alternative procedure, asolution phase sandwich assay may be used with labeled polynucleotideprobes, and the methods for the preparation of such probes are describedin U.S. Pat. No. 4,820,630, issued Apr. 11, 1989, which is incorporatedherein by reference.

Therefore, the probes described below are preferably applied to thedetection of meningitis by using them in combination to detect andidentify what bacteria are present in a sample of cerebrospinal fluid.All of the probes described below, as well as additional probes, can bearranged in a reverse dot blot format, as described by Saiki et al.,(supra.) Each of the probes is immobilized as a separate dot on a solidsupport such as a nylon membrane or microtiter plate. The amplified DNAis hybridized to each of the probes at the same time in an aqueoussolution. The pattern of the signals from each of the dots (i.e.,probes) indicates the identity of the target DNA. Accordingly, uponamplification of the target region (preferably by PCR), and applicationof the panel of probes described herein, hybridization of one or more ofthe probes in the panel (including the universal probe when applied toCSF) will result in a positive signal and the positive identification ofthe bacterial species present as either Listeria monocytogenes, E.coli/enteric bacteria, Haemophilus influenzae, Neisseria meningitidis,Streptococcus pneumoniae, S. agalactiae, Staphylococcus epidermidis,Propionibacterium acnes, Propionibacterium species, Bacillus species,coagulase-negative Staphylococci, Corynebacterium species,Staphylococcus aureus, or a bacterium which does not react with any ofthe more specific probes.

Those skilled in the an will also be aware of the problems ofcontamination of a PCR by the amplified nucleic acid from previousreactions and non-specific amplification. Methods to reduce theseproblems are provided in PCT patent application Ser. No. 91/05210, filedJul. 23, 1991, incorporated herein by reference. The method allows theenzymatic degradation of any amplified DNA from previous reactions andreduces non-specific amplification. The PCR amplification is carried outin the presence of dUTP instead of dTTP. The resulting double-strandeduracil-containing product is subject to degradation by uracilN-glycosylase (UNG), whereas normal thymine-containing DNA is notdegraded by UNG. Adding UNG to the amplification reaction mixture beforethe amplification is started degrades all uracil-containing DNA thatmight serve as target. Because the only source of uracil-containing DNAis the amplified product of a previous reaction, this method effectivelysterilizes the reaction mixture, eliminating the problem ofcontamination from previous reactions (carryover). UNG itself isrendered temporarily inactive by heat, so the denaturation steps in theamplification procedure also serve to inactivate the UNG. Newamplification products, therefore, though incorporating uracil, areformed in an effectively UNG-free environment and are not degraded.

Also within the scope of the present invention are amplification anddetection kits for use in carrying out any of the aforementionedamplification and detection processes. The diagnostic kits include thepolynucleotide probes and the primers in separate containers. Either ofthese may or may not be labeled. If unlabeled, the ingredients forlabeling may also be included in the kit. The kit may also contain othersuitably packaged reagents and material needed for the particularhybridization protocol, for example, standards, and/or polymerizingagents, as well as instructions for conducting the test.

In use, the components of the PCR kit, when applied to a nucleic acidsample, create a reagent mixture which enables the detection andamplification of the target nucleic acid sequence. The reagent mixturethus includes the components of the kit as well as a nucleic acid samplewhich contains the polynucleotide chain of interest. The teachings ofthe references cited in the present application are incorporated hereinby reference.

A variation of this invention is to use an alternate method of producingthe amplified target region. For example, the TAS amplification system,(Kwoh et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-177) and itsmodification, SSSR (Guatelli et al., 1990, Proc. Nat. Acad. Sci. USA 87:1874-1878) is a method for amplifying RNA or DNA templates using cyclesconsisting of a cDNA step to produce a cDNA copy of an RNA template, andan RNA transcription step to increase the copy number of the cDNA or DNAtemplate. This method, like PCR, employs two oligonucleotide primerswhich hybridize to opposite strands of the target region and flank thetarget region. The universal bacterial primers described herein may,with minor modifications (the addition of RNA polymerase promotersequences at the 5' end of one of the primers), be used in a TAS or SSSRamplification system. The subsequent step of the assay, detection by theoligonucleotide probes described herein, may be carried out essentiallyas described above for the PCR-based assay or may be done using abead-based sandwich hybridization system (Kwoh et al.).

The nucleotide sequence data described herein can also provide specificdetection of bacterial species when used in other nucleic acid-basedassays. For example, the nucleotide sequence information discovered forS. pneumoniae and S. agalactiae indicated that there is a singlebase-pair mismatch between these two organisms in the region of the S.pneumoniae probe RDR224 (SEQ ID No. 23). This mismatch could be used ina ligase chain reaction system to provide discrimination between thesetwo organisms in a clinical sample (Wu and Wallace, 1988, Genomics 4:560-569). The ligase chain reaction involves the use of two sets ofoligonucleotide primers. Each primer within the set is complementary tothe other. The different sets of primers are located directly adjacentto each other along the template. A single base pair mismatch in betweenthe two sets of primers disrupts the reaction, whereas a perfect matchbetween the primer sets and the template results in targetamplification. In another example, the sequence of probes describedherein could be used to design corresponding probes in a signalamplification system such as the Q beta replicase system (Kramer andLizardi, 1989, Nature 339: 401-402, and Lomeli et al., 1989, Clin. Chem.35: 1826-1831). This system involves an RNA probe containing thespecific probe sequence inserted into the MDV-1 variant of the Q-betaRNA genome. The RNA probe is replicated using Q-beta replicase,producing up to 10¹² molecules per reaction, after hybridization of theprobe to the sample to be assayed.

By way of further specificity, the following probe and primer nucleotidesequence data is provided:

Primer DG74 (SEQ ID No. 26) corresponds to the complement of nucleotidebase numbers 1522-1540 in the E. coli 16S ribosomal RNA gene asspecified in Neefs supra.

Primer RW01 (SEQ ID No. 27) corresponds to nucleotide base numbers1170-1189 in the E. coil. 16S ribosomal RNA gene as specified in Neefssupra.

Further, FIG. 1 provides a description of nucleotide sequences isolatedas described below, which can be used to design and formulate probes andprimers corresponding to fragments or subsequences thereof or theircomplements.

Oligonucleotide probes for various bacterial species are shown in FIG.2.

The following examples are intended to be illustrative of the variousmethods and compounds of the invention.

EXAMPLE 1 Methods Used to Obtain Sequence Data and Design Probes

A. DNA Sequencing Protocol

In order to obtain the DNA sequence from the bacterial species desired,it was necessary to (1) amplify the amount of the target region presentand then (2) isolate the individual DNA strands (single-stranded DNA)for use in the sequencing reactions.

DNA was prepared from cell pellets of various bacterial species bytreatment with lysozyme, SDS and proteinase K according to the method ofSilhavy et al. (Silhavy, T. J., M. L. Berman, and L. W. Enquist, 1984,Experiments with gene fusions, pages 137-139. Cold Spring HarborLaboratory, Cold Spring Harbor, N.Y.). Bacterial strains were obtainedin the form of cell pellets (frozen or non-frozen). The concentration ofDNA in the preparations was estimated by gel electrophoresis. Ten ngamounts of DNA were amplified in two different ways. One set of PCRreactions utilized a biotinylated PL06 (5'-GGTTAAGTCCCGCAACGAGCGC[SEQ IDNo. 46]) and nonbiotinylated DG74 (SEQ ID No. 26) whereas the other setof reactions utilized nonbiotinylated PL06 and biotinylated DG74 (SEQ IDNo. 26). The conditions used for the amplifications were as described inExample 2, except that the number of cycles was 25 and an annealingtemperature of 60° C. rather than 55° C. was used for DNA from S.agalactiae, S. epidermidis, S. pneumoniae, and L. monocytogenes, S.aureus, and S. pyogenes.

For the preparation of single-stranded DNA to use as a template in thesequencing reactions, two methods were used. One method was based on theuse of streptavidin agarose beads and was used for the sequencing of S.agalactiae and S. pneumoniae (Mitchell and Merrill, 1989, AnalyticalBiochemistry 178: 239-242). The other was based on the use ofstreptavidin linked to magnetic beads and was used for sequencing of N.meningitidis, S. epidermidis, S. aureus, and S. pyogenes (Bowman andPalumbi, Molecular Evolution: Producing the Biochemical Data, [A Volumeof Methods in Enzymology], Zimmer et al., eds. [in press]). In the firstmethod, 90 μl of the amplified DNA was combined with 200 μl ofstreptavidin agarose slurry (Bethesda Research Laboratories, catalog#59442SA) in a 2.0 ml microcentrifuge tube. The mixture was incubated atroom temperature for 30 minutes or more, mixing frequently or rotatingon a Labquake apparatus. The mixture was spun in an Eppendorf 5415microfuge for 10 seconds at speed setting #1. The supernatant wasremoved. The pellet was washed with 500 μl of TE (10 mM Tris-HCl, 1 mMEDTA, pH 8.0) by shaking gently or Labquaking for 1 minute. The mixturewas spun in the microfuge for 10 seconds at speed setting #1. Thesupernatant was removed. The TE wash was repeated once more. Freshlydiluted 0.2M NaOH (150μl) was added to each pellet The tube wasincubated for 6 minutes at room temperature, Labquaking or tapping thetube frequently. The mixture was spun in a microfuge at setting #10 for1 minute. The supernatant was removed and retained. The NaOH treatmentwas repeated and the two supernatants combined. Two hundred μl ofammonium acetate (5M, pH 6.8) was added to the combined supernatants andmixed well. The tubes were spun in the microfuge for 5 minutes atsetting #13 to pellet any debris. The supernatant (480 μl) wastransferred to a new microcentrifuge tube, leaving 20 μl left in thetube. This pelleting step was repeated once more, with 460 μl beingtransferred to the top of a centricon-100 tube and 20 μl being left inthe microfuge tube.

In the second method, the PCR product was first treated to an optionalprespin in a Centricon-100 tube to purify away the amplificationprimers. Ninety-five to 100 μl PCR product plus 1.9 ml glass distilledH₂ O were combined in the top of a Centricon-100 tube and spun for 25minutes at 3000 rpm in an SA-600 or comparable rotor. The product wasthen backspun into a retentate tube in a tabletop centrifuge andcombined in a 2.0 ml microcentrifuge tube with 100 μl streptavidin (SA)magnetic bead slurry (Promega, catalog #Z5241). The mixture wasincubated at room temperature for 30 minutes or more, mixing frequentlyor rotating on a Labquake apparatus. The mixture was pulse-spun at topspeed in the microfuge extremely briefly to remove the liquid and beadsfrom the lid of the tube without spinning the beads to the bottom of thetube. The mixture was gently tritterated with a pipet and the tube wasthen placed next to a small magnet until the beads collected on the sideof the tube. Holding the magnet alongside the tube, the liquid was thenpipetted away from the beads without disturbing them. The beads werethen washed with 500 μl TE, by shaking gently or Labquaking for 1minute. The tube was pulse-spun, the sample tritterated and exposed tomagnet, and the liquid removed from the beads. The TE wash was repeatedonce more. Freshly diluted 0.2M NaOH (150 μl) was added to each tube ofbeads. The tube was incubated for 6 minutes at room temperature,Labquaking or tapping the tube frequently. The tube was pulse-spun, thesample tritterated and exposed to magnet, and the liquid removed fromthe beads and added to 200 μl ammonium acetate (5M, pH 6.8) already inthe top of a centricon-100 tube. The NaOH treatment was repeated and thesecond supernatant added to the centricon tube as well.

The remainder of the procedure was the same for both methods. Thesolution was rinsed two times with 2 ml of glass-distilled water byspinning the centricon-100 tube at 3000 rpm for 30 minutes in a SorvallSS34 or SA600 rotor. An additional spin with another 2 ml of water at3000 rpm for 45 minutes was done. The Centricon-100 top reservoir wascapped and inverted, 20 μl of water were added and the tube was backspunfor a short time at 1000 rpm. The retained solution was transferred to amicrofuge tube and dried in a Speed Vac evaporator for 1 hour at themedium heat setting. The single-stranded DNA was resuspended in 10 μl H₂O. Four to 7.5 μl of the solution was used in a Sequenase sequencingreaction according to manufacturer's instructions (United StatesBiochemical Corporation, Cleveland, Ohio)

B. Characterization of Oligonucleotide Probes

Oligonucleotide probes for each of the species to be detected were basedon two sources of information: (1) the sequence data described in FIG. 2and (2) data in Genbank. In addition, the nucleotide sequence forListeria monocytogenes was determined experimentally and is identical inthe probe region to the sequence published by Collins et al., 1991,International Journal of Systematic Bacteriology 41: 240-246. Each ofthe candidate probes was evaluated using the following steps. First, thenucleotide sequence within the 370 bp region bounded by amplificationprimers RW01 (SEQ ID No. 27) and DG74 (SEQ ID No. 26) obtained for eachspecies was compared to that of a panel of other species, consisting ofspecies on the meningitis panel (N. meningitidis, S. pneumoniae, S.agalactiae, H. influenza, L. monocytogenes, and E. coli), some closelyrelated species (such as Pasteurella multocida, Neisseria gonorrhoeae,N. denitrificans, and Kingella indologenes), and some species consideredcontaminants (such as Bacillus substilis, B. brevis). In this manner,regions where differences in the sequence occurred could be found. Mostof the differing regions were within "variable regions 8 and 9 " whichhave been characterized in the scientific literature previously(corresponding approximately to positions 1236-1300 and 1409-1491,respectively, in the E. coli 16S rRNA gene). From the regions ofsequence variability, candidate 25 base pair probes were chosen.

Second, each oligonucleotide was examined for self-complementary(ability to form base pairs with itself) using a computer program calledOLIGO, (National Biosciences, Hamel, Minn.). The position of theoligonucleotide probe was chosen to minimize the formation of secondarystructure where it was possible to do so while still maintaining thedesired specificity. For example, self-complementarity of more than 6consecutive bases was avoided.

Third, the candidate probes were compared with the correspondingnucleotide sequence of more phylogenetically diverse species listed inGenbank to find the probes that would not detect other species. In caseswhere the probe was capable of hybridizing to other species, itslocation was chosen to minimize hybridization to other pathogenic orcontaminant species found in CSF as much as possible. For example,mismatches of a probe to a species which could potentially give across-reaction were centered within the probe to minimize the crossreaction.

EXAMPLE 2 Methods for Specificity Testing of Probes

A. PCR Amplification

PCR amplification of bacterial DNA was accomplished as follows.

A standard PCR 2× mix was made containing the following for amplifying atarget sequence for bacteria:

    ______________________________________                                        10× standard PCR buffer                                                                           10.0   μl                                        50 mM MgCl.sub.2          1.0    μl                                        dNTP's (2.5 mM total dNTP's)                                                                            2.5    μl                                        primer RWO1 (SEQ ID No. 27) (50 μM)                                                                  1.0    μl                                        primer DG74 (SEQ ID No. 26) (50 μM)                                                                  1.0    μl                                        H.sub.2 O                 34.0   μl                                        Taq DNA polymerase (5 U/μl)                                                                          0.5    μl                                        ______________________________________                                    

The 10× standard PCR buffer contains:

100 mM Tris-HCl, pH 8.3

500 mM KCl

15 mM MgCl

0.1% (w/v) gelatin

Fifty μl of a bacterial DNA sample was mixed together with 50 μl of thePCR 2× mix.

The reaction mixture was placed in a 0.5 ml microfuge tube and the tubewas placed in a thermal cycler manufactured by Perkin-Elmer. A two-stepPCR cycle was used and the thermocycler was set as follows:

1. Time delay file--5 minutes at 95° C.

2. Thermocycle file--95° C. for 25 seconds 55° C. for 25 seconds for 25cycles

3. Time delay file--10 minutes at 72° C.

B. Detection of Amplified Products

After the amplification reaction was complete, 5 μl of the 100 μl PCRreaction was mixed with DNA dye buffer (1 μl of 50% sucrose, 10 mM Tris,pH 7.5, 1 mM EDTA, 1.0% SDS, 0.05% bromphenol blue. Alternatively, 0.6μl of 25% Ficoll, 0.5% bromphenol blue, 0.5% xylene cyanol, 0.5% orangeG, 5 mM EDTA, pH 8.0, 0.5% SDS can be used). The sample was loaded ontoa 2% Nusieve agarose, 0.5% Seakem agarose, 1× TBE (45 mM Tris-borate, 1mM EDTA) gel. After running the bromphenol blue or orange G dye front tothe bottom of the gel, the gel was stained with ethidium bromide (5μg/ml), washed in water and photographed under UV light using a Polaroidcamera and an orange filter.

The size of the PCR product was approximately 370 bp.

C. Transfer of Amplified DNA to Nylon Membrane

After photography of the gel, the gel was soaked in 0.25N HCl for 10minutes at room temperature. The gel was then soaked in solution of 0.5NNaOH, 1.5M NaCl for 15 minutes. The gel was then soaked in a solution of1M Tris, pH 7.5, 1.5M NaCl for 15 minutes. The gel was then rinsed in20× SSPE.

DNA was then transferred to a nylon membrane (Pall Biodyne) either dryor presoaked in water by one of two ways: (1) vacuum transfer using aStratagene Stratavac vacuum blotter or (2) capillary transfer by themethod of Southern.

After transfer, DNA was fixed to the membrane using UV light in aStratagene Stratalinker.

D. Radioactive Labeling of Oligonucleotide Probes

The oligonucleotide probes were labeled using T₄ polynucleotide kinasein one of the following reaction mixes:

    ______________________________________                                                         Mix 1     Mix 2                                              ______________________________________                                        .sup.32 P ATP    10      μl 6.0    μl                                   10× kinase buffer                                                                        2.5     μl 2.5    μl                                   oligonucleotide (10 μM)                                                                     2.0     μl 1.0    μl                                   H.sub.2 O        8.5     μl 14.5   μl                                   T4 polynucleotide kinase                                                                       2.0     μl 1.0    μl                                   ______________________________________                                    

10× kinase buffer contains:

500 mM Tris, pH 8

100 mM MgCl₂

50 mM DTT

The kinase reaction mixture was incubated for 30 minutes at 37° C. 5.6of 0.25M EDTA and 169.4 μl of H₂ O were added to stop the reaction. Thismixture was loaded onto a 1.0 ml capacity column of Biogel P4 and spunin a tabletop centrifuge for 5 minutes at 2,500 rpm to separate thelabeled oligonucleotide from the unincorporated radioactivity. 1 μl ofthe eluate from the column was counted in a scintillation counterwithout added scintillation fluid (Cerenkov counting) to obtain anestimate of the level of incorporation of radioactivity. A volume givingapproximately 1-2×10⁶ cpm was used for each blot in the subsequenthybridization.

E. Hybridization of Probes with DNA

The DNA blots were prehybridized in a mixture of 5× SSPE, 0.5% SDS at60° (1×SSPE=0.18M NaCl, 10 mM NaPO₄, pH 7.4, 1 mM EDTA). The labeledoligonucleotide probe was added to 10.0 ml of 5× SSPE, 0.5% SDS andmixed. The solution was added to the plastic bag containing the drainedpresoaked blot. The blot was incubated for 1 to 18 hours at 60° C.

The blot was removed from the plastic bag and placed in a solution of2×SSPE, 0.1% SDS and washed for 10 minutes at room temperature. The blotwas then washed in a solution of 3M tetramethylammonium chloride(TMACl), 50 mM Tris, pH 8 and 0.2% SDS for 10-20 minutes at roomtemperature, followed by an additional wash for 10 minutes at 62°-64° C.

The blot was wrapped in Saran wrap and placed in a X-ray film holderwith a sheet of Kodak XAR-5 X-ray film with or without an intensifyingscreen for 1 to 72 hours at -70° C.

EXAMPLE 3 Results of Specificity Testing of Oligontlcleotide Probes

Each of the probes was tested against PCR products from various bacterialisted in FIG. 3. The bacteria selected for testing represent two typesof species (1) those of which can be found in cerebrospinal fluid (CSF)either as pathogens or contaminants or (2) those which are closelyrelated to the first type of species. The methods used (for detection ofamplified products, transfer of the amplified DNA to nylon membranes,radioactive labeling of oligonucleotide probes and hybridization ofprobes to the membrane) were as described in Example 2 with temperaturesused for the washes in TMACl (tetramethylammonium chloride) ranging from62° to 66° C. In the hybridization results shown in FIG. 3, thetemperature was 66° C. for all probes.

For some of the organisms to be detected, it was unexpectedly found thata number of different probes had to be evaluated before a probe withsatisfactory characteristics could be defined. The Listeriamonocytogenes probe RDR232, (SEQ ID No. 42)5'-AGGGTAACCTTTATGGAGCCAGCCG-3', at 62° C. also hybridized to Bacilluscereus and slightly to S. salivarius; whereas the L. monocytogenes probeRDR230 (SEQ ID No. 11) did not hybridize to either of these strains at62° C., indicating a greater degree of specificity for RDR230 (SEQ IDNo. 11). Streptococcus agalactiae probes RDR255, (SEQ ID No. 39)5'-CCTTTTAGGAGCCAGCCGCCTAAGG-3', and RDR257, (SEQ ID No. 40)5'-CCTTAGGCGGCTGGCTCCTAAAAGG-3', were found to be unsatisfactory due topoor hybridization signals at 66° C., even though the hybridizationsignals at 62° C. and 64° C. were satisfactory. In addition, at 64° C.,S. agalactiae probe RDR255 (SEQ ID No. 39) detected B. subtilis, B.cereus and S. salivarius. S. agalactiae probe RDR254, (SEQ ID No. 38)5'-TAACCTTTAGGAGCCAGCCGCCTA-3', detected B. subtilis in addition to S.agalactiae at 66° C. S. agalactiae probe RDR306 (SEQ ID No. 20) gavegood hybridization signals at 66° C. and did not hybridize to B.subtilis, B. cereus or S. salivarius. Probe RDR324,5'-CGGTTTCGCTGACCCTTTGTATTGT-3' (SEQ ID No. 41), for the contaminantspecies Staphylococcus epidermidis did not hybridize well at 66° C.;moreover, at lower temperatures (62° C., 64° C.) it hybridized to S.aureus, a pathogen. By comparison, S. epidermidis probe RDR325 (SEQ IDNo. 6) did not hybridize to S. aureus at 62° C. or 64° C.

As shown in FIG. 3, each of the probes selected showed a high degree ofspecificity for the bacterial species tested. In particular, RDR230 (SEQID No. 11) (Listeria monocytogenes), RDR140 (SEQ ID No. 9) (E.coli/enteric bacteria), RDR125 (SEQ ID No. 10) (Haemophilus influenzae),RDR328 (SEQ ID No. 15). (Propionibacterium acnes) and RDR325 (SEQ ID No.6) (Staphylococcus epidermidis) detected only the intended species amongup to 39 different species tested. The Streptococcus pneumoniae probeRDR224 (SEQ ID No. 23) hybridized to S. mitis and partially to S.anginosus, S. milleri. S. sanguis, and S. intermedius in addition to S.pneumoniae. The cross-reacting species are found in the oral cavity andare not common causes of meningitis. The Streptococcus agalactiae probeRDR306 also hybridized to 4 other Streptococci (S. equi, S. group G, S.pyogenes and S. dysgalactiae). Some of these other Streptococci arepathogenic, but are infrequently found in CSF. The Neisseriameningitidis probe RDR307 (SEQ ID No. 13) hybridized to Neisseria siccain addition to N. meningitidis. N. sicca is found in the nasopharynx,saliva and sputum of humans and is not frequently found in CSF. For allof the cross-reacting probes, it is believed that minor and readilydeterminable modifications confer the required specificity.

EXAMPLE 4 Format II Detection Method

The following demonstrates a preferred embodiment of the invention. ACSF sample suspected of containing nucleic acid from a bacterium isextracted and is amplified with universal bacterial primers RW01 (SEQ IDNo. 27) and DG74 (SEQ ID No. 26) as described in Example 2 or 7.

The probes are the same in sequence as those described herein, but aremodified to contain 100 to 150 T residues at the 5' end. The probes arefixed to a nylon membrane in a dot blot format by a method similar tothat of Saiki et al., 1989. Each dot corresponds to a single probe (oneeach for the universal bacterial probe, N. meningitidis probe, L.monocytogenes probe, E. coli/enteric bacteria probe, S. pneumoniaeprobe, S. agalactiae probe, H. influenzae probe, S. epidermidis probe,P. acnes probe, Bacillus species probe, Corynebacterium species probe,etc.) One strip or set of strips is used for each sample to be tested.The strips are placed in the wells of a plastic tray and 3 to 5 ml ofhybridization solution is added (5× SSPE, 0.5% w/v SDS, preheated to 37°C.).

The amplified DNA is denatured by incubation at 95° C. in the heat blockof a thermal cycler for 3 to 10 minutes. The tube with the amplified DNAis removed from the heat block and 25-35 μl is immediately removed andadded to the well containing the dot blot strip. The tray is incubatedat 50° C. to 65° C. in a shaking water bath at 50-90 rpm for 20 to 30minutes. After hybridization, the solution is aspirated from the well ofthe tray. The strips are rinsed in 5 ml of wash solution (2.5× SSPE,0.1% w/v SDS, preheated to 37° C.) at room temperature for 1 to 5minutes. The wash solution is aspirated and 5 ml more is added. Thestrips are incubated at 50° to 65° C. for 10 to 15 minutes at 50 rpm.The solution is aspirated and 5 ml of wash solution is added. The stripsare incubated for 1 to 5 minutes at room temperature. The wells areaspirated. Five ml of hybridization solution is added and thenaspirated.

Three ml of a mixture of 3.3 ml hybridization solution and 27 μl ofenzyme conjugate (Amplitype kit [developed and manufactured byHoffmann-La Roche and marketed by Perkin Elmer] or suitable substituteprepared in a glass flask) per strip is added to each strip andincubated for 30 minutes at room temperature at 50 rpm. Followingaspiration wash solution (5 ml) is added and incubated for 5 to 10minutes at room temperature. Repeat the wash. Citrate solution (0.1Msodium citrate, pH 5.0), 5 ml, is added and incubated at roomtemperature for 5 minutes. A mixture of 5 ml citrate buffer 5 μl of 3%hydrogen peroxide, and 0.25 ml TMB (Amplitype kit) is added (4 to 5 mlper strip) and incubated for 20 to 30 minutes in the dark to allow colordevelopment. To stop color development, the strips are rinsed indistilled water three times. The strips are photographed in visiblelight against a dark background using Polaroid type 55 or 57 film and anorange filter.

The invention can also be practiced in a microtiter plate format, inwhich the probes are affixed to the bottom of the wells of a microtiterplate via a thioether linkage to bovine serum albumin (Barone et al.,1991, Abstracts Am. Soc. Microb., page 361).

EXAMPLE 5 Additional Considerations in Design of Probes

Two characteristics are essential for the probes included in themeningitis panel. The first characteristic is the inclusivity of theprobe. Probes for a given species preferably detect all strains of thespecies, including strains of various serotypes that have beenclassified by their reaction with specific antibodies. For example, theL. monocytogenes probe reacts with the following serotypes of L.monocytogenes--serotypes 1/2a, 1/2b, 1/2c, 3b, 4b. These serotypes arethe ones most commonly found in clinical samples. Most preferably, an L.monocytogenes probe reacts with all serotypes of L. monocytogenesincluding 1/2a, 1/2b, 1/2c, 3a, 3b, 3c, 4a, 4b, 4c, 4d, 4e, and 7.

The second characteristic is the exclusivity of the probe. Probes for agiven species should not detect other species. Based on DNA sequencecomparison, the existing L. monocytogenes probe may react with thefollowing Listeria species--L. innocua, L. welshimeri, L. seeligeri, L.ivanovii. These other Listeria species are very rarely found in clinicalspecimens. A preferred probe does not react with any listeria speciesother than L. monocytogenes.

In practice, although it is preferred, it is not essential to achievethe desired inclusivity and exclusivity of a given probe. In thesecases, the major considerations in determining the usefulness of a probein the meningitis assay are: (1) for inclusivity--the probe is able todetect most species found in CSF as pathogens or contaminants and (2)for exclusivity--species that the probe does detect are not commonlyfound in CSF.

The same considerations discussed above would apply to the other probeson the panel. Hence, the preferred characteristics of the probes are asfollows:

Haemophilus influenzae probe

Inclusivity--all strains of H. influenzae detected

Exclusivity--no species other than H. influenzae detected

S. pneumoniae probe

Inclusivity--all strains of S. pneumoniae detected

Exclusivity--no species other than S. pneumoniae detected

The S. pneumoniae RDR224 (SEQ ID No. 23) probe gave a weak reaction withthe following species: S. mitis, S. anginosus, S. milleria, S. sanguis,and S. intermedius. Probe RDR462 (SEQ ID No. 24) was designed to improvethe specificity of detection. This probe does not cross react with S.anginosus, S. millei, S. sanguis, or S. intermedius.

E. coli/enteric bacteria probe

Inclusivity--detects all strains of all species of enteric bacteria

Exclusivity--no species other than enteric bacteria detected

Neisseria meningitidis probe

Inclusivity--detects all serotypes and strains of N. meningitidis

Exclusivity--no species other than N. meningitidis detected

Probe RDR307 (SEQ ID No. 13) was found to not react with the followingserotypes of N. meningitidis--serotypes B, C, Y, and W135. To improvethe range of detection of this probe, the new probe COR28 (SEQ ID No.14) was designed and tested. Probe COR28 (SEQ ID No. 14) reacts with thestrains of serotypes A, C, B, Y, and W135 of N. meningitidis that weretested, but also reacts with the following Neisseria species--N.gonorrhoeae and N. gononhoeae kochii. Because the latter species israrely found in CSF, COR28 (SEQ ID No. 14) is considered a suitableprobe for detecting bacteria in CSF.

S. agalactiae probe

Inclusivity--detects all strains of S. agalactiae

Exclusivity--no species other than S. agalactiae detected

Corynebacterium species probe

Inclusivity--detects all strains and species of Corynebacterium,including uncharacterized species colonizing human skin

Exclusivity--no species other than Corynebacterium detected

S. epidermidis and coagulase-negative Staphylococcus probes

Inclusivity--preferably a single probe would detect all strains andspecies of coagulase-negative Staphylococci

Exclusivity--no species other than coagulase-negative Staphylococcidetected

The S. epidermidis probe RDR325 (SEQ ID No. 6) detects S. epidermidis, acoagulase-negative Staphylococcus species, but does not detect othercoagulase-negative Staphylococci. DNA sequence analysis of the followingstrains was used to design an improved probe: S. auricularis, S.saccharolyticus. The sequence analysis indicated that it was notpossible to design a single probe that would detect all three of thecoagulase-negative Staphylococcus species. A separate probe, RDR512 (SEQID No. 4), was designed which detects S. auricularis and S.saccharolyticus. Further specificity testing of RDR325 (SEQ ID No. 6)and RDR512 (SEQ ID No. 4) indicated that RDR325 (SEQ ID No. 6) alsodetects S. haemolyticus and RDR512 (SEQ ID No. 4) detects S. capitis butneither probe detects other coagulase negative Staphylococcus speciessuch as S. cohnii, S. hominis, S saprophyticus, and S. warneri.

S. aureus probe

Inclusivity--all strains of S. aureus detected

Exclusivity--no species other than S. aureus detected

Bacillus species probe

Inclusivity--all strains and species of Bacillus detected

Exclusivity--no species other than Bacillus detected

Propionibacterium species probe

Inclusivity--all strains and species of Propionibacterium detected

Exclusivity--no species other than Propionibacterium detected

Probe RDR328 (SEQ ID No. 15) was found to not react with the followingPropionibacterium species--P. avidum, P. granulosum, P. lymphophilum.Because P. avidum and P. granulosum are known to colonize human skin,they should be detected by a Propionibacterium species probe. ProbeRDR514 (SEQ ID No. 17) was designed and tested as an improvement ofRDR328 (SEQ ID No. 15), based on DNA sequence information for theseother Propionibacterium species. This probe was found to react with P.acnes, P. avidum, P. granulosum, and P. lymphophilum.

Changes in the detection scheme necessitate reoptimization of probesequences and hybridization conditions as described herein. For example,probes that were suitable in a Southern blot format using TMACl (resultsdescribed in Example 3) were re-evaluated for specificity when used inthe reverse dot blot format (method described in Example 4). In order toachieve the required specificity, the S. pneumoniae, Bacillus species,and S. aureus probes were modified by making them shorter by 2, 4, and 3bases, respectively. The coagulase-negative Staphylococcus probe wasmodified by introducing a mismatch in the center. The Corynebacteriumspecies probe was modified by using its reverse complement. A new regionwas used for the Propionibacterium species probe. The S. epidermidisprobe was shortened by two bases and the reverse complement was used. Adifferent region was chosen for the modified S. agalactiae probe. VP109(SEQ ID No. 21) was used in the reverse dot-blot format; its reversecomplement, KG0001 (SEQ ID No. 22), was used in Southern blot format.These modifications were found to improve the specificity of the probesin reverse dot blot format as demonstrated in FIG. 4.

Additional modifications may improve the performance of probes whenusing dUTP in the amplification mix. It was found that the signaldetected for certain PCR products with certain probes was reduced whendUTP was substituted for TTP in the amplification mix. One possibleexplanation is that the hybridization efficiency of the U-containing PCRproducts is reduced relative to that of T-containing PCR products. Forthe detection of L. monocytogenes, a probe selected from the followingcompositions may give improved performance.

This is the reverse complement of RDR230 (SEQ ID No. 11) SEQ ID No. 325'-ACT GAG AAT ACT TIT ATG GGA TTA G-3'

These are located in a different region of the 16S gene and are reversecomplements of each other.

SEQ ID No. 33 5'-AGG GTA ACC TTT ATG GAG CCA GCC G-3'

SEQ ID No. 34 5'-CGG CTG GCT CCA TAA AGG TTA CCC T-3'For the detectionof S. agalactiae, a probe of the following composition may represent animprovement (this is probe KG0001 (SEQ ID No. 22) with 2 bases removedfrom the 5' end).

SEQ ID No. 35 5'-ATCTCTTAAAGCCAATCTCAGTF-3'

This probe VP109 (SEQ ID No. 21) shortened by 2 bases at the 3' end.

SEQ ID No. 36 5'-AACTGAGATTGGCTTTAAGAGAT-3'

EXAMPLE 6 Preparation of Low-DNA Taq Polymerase

To increase sensitivity of the present methods, it may be desirable touse amplification cycle numbers higher than 25 (e.g., 26-40). However,the extreme sensitivity of such a reaction using the universal primersdisclosed may lead to artifactual results due to amplification ofresidual DNA in commercial reagents. For high cycle number the followingprocedure eliminates DNA contamination in the agent for polymerization.

Equipment Required

Biorad Econo-pac Q cartridge; Biorad catalog #732-0021

Sterile disposable 50 mL polypropylene tubes; Corning catalog #25330-50

HPLC/FPLC flow adaptors; Biorad Catalog #732-0111/732-0112.

General laboratory equipment

Peristaltic pump (flow rate 0.5-2 mL capability)

Reagents Required

Formulation buffer: 20 mM Tris, 0.1M KCl, 0.5% NP40, 5% Tween-20, 1 mMDTT, 0.1 mM EDTA, 50% glycerol, pH 8

Econo-pac Q wash buffer: 200 mM Tris/1M KCl , pH 8.8

0.5N Acetic acid

1.0N Sodium hydroxide

Sterile Glass distilled water

10% bleach

70% Ethanol

Procedure

A. Preparation of Laminar Flow Hood, Peristaltic Pump, and CartridgeFittings

1. Wipe down the hood with 10% bleach.

2. Install the peristaltic pump with tubing and cartridge HPLC/FPLC flowadaptors.

3. Install the column support stand and clamps into the hood.

4. Turn on UV lamp for 30 minute to irradiate surfaces.

5. Rinse pump tubing with 20 mL 70% Ethanol at a flow rate of 1 mL/min.

6. Rinse tubing with 50 mL sterile glass distilled water at a flow rateof 1 mL/min.

7. Discard the wash fluid.

B. Washing the Econo-Pac O Cartridge

Note: All washes are to be performed using the peristaltic pump at aflow rate not exceeding 2 mL/min. Discard all wash fluid after use. Alloperations carried out in the hood.

1. Attach cartridge to the column support stand.

2. Connect tubing to the cartridge by the flow adaptors.

3. Wash cartridge with 20 mL sterile GD water.

4. Wash cartridge with 50 mL 0.5N acetic acid.

5. Wash cartridge with 50 mL 1.0N sodium hydroxide.

6. Wash cartridge with 50 mL Econo-pat Q wash buffer.

7. Wash cartridge with 50 mL Formulation buffer.

8. Calibrate flow rate to 0.5 mL/min.

C. Loading and Collecting Ampli Taq® DNA Polymerase

1. Remove Ampli Taq® DNA polymerase stock from -20° C. freezer. Allow tothaw at room temperature for 30 minutes.

2. In the hood, add 100 mL of AmpliTaq® DNA polymerase to a sterile,heat treated 250 mL flask.

3. Replace the stock AmpliTaq® DNA polymerase into the freezer.

4. Load AmpliTaq® onto the cartridge at a flow rate of 0.5 mL/min.

5. Collect 5 mL into a tube, then switch to a clean 50 mL tube. Discardthe 5 mL aliquot.

6. Collect 25 mL aliquots of AmpliTaq® DNA polymerase into sterile 50 mLtubes.

EXAMPLE 7 A Preferred Method for Analysis of Clinical Samples

Two modifications of the amplification conditions in Example 2 arepreferred when testing clinical samples. First, modifications whichgreatly reduce the possibility of carry-over contamination are used. Thenucleotide dUTP is substituted for TTP in the amplification mix, anduracil-N-glycosylase (UNG) is added to the amplification mix. Under theappropriate conditions of concentration and incubation, thesemodifications degrade any U-containing PCR product that may contaminatethe reaction.

The second modification is to treat the amplification reagents to reducethe level of contaminating bacterial DNA present This allowsamplification cycle numbers higher than 25 to be used for increasedsensitivity. The 10× Taq buffer (100 mM Tris-HCl, pH 8.3, 500 mM KCl) isautoclaved and sterilely dispensed. Eight mM MgCl₂ is autoclaved andsterilely dispensed. Water is ultrafiltered and autoclaved. TE buffer(10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0) is autoclaved.

The solution of dNTPs (dATP, dGTP, dCTP, dUTP) is filtered through aCentricon-30 filter (Amicon catalog number 4208). The C-30 filtrate cupand cap are autoclaved and the filter unit is soaked in 10% bleach for 1hour. The filters are thoroughly rinsed in autoclaved ultrafilteredwater. The filters are then spun with 400 μl of autoclaved ultrafilteredwater. The dNTP solution is centrifuged through the treated C-30 filtertraits once for 30 minutes at 5000 xg in a fixed angle rotor.

The primers at 200 uM concentration are filtered through Millipore M-100PTHK filters (catalog number UFC3THYK00). The Eppendorf tube part of thefilter unit is autoclaved and the filtrate cup is soaked in 10% bleachfor 1 hour. The filters are thoroughly rinsed in autoclavedultrafiltered water. The filters are then spun with 400 μl of autoclavedultrafiltered water at 5000 xG for 3 minutes in an Eppendorf microfuge.Each primer is successively filtered 4 times, each time through a cleanfilter for 3 minutes at 5000× g. The primers are diluted 1:250 for aOD₂₆₀ reading. Primer concentration is adjusted using autoclaved TEbuffer to 20 um.

The 4× PCR mix is made up as follows:

400 μl 10X Taq buffer

40 μl 100 mM dNTP mix or 400 μl of a mixture of equal volumes of 10 mMof each dNTP

80 μl of 20 uM RW01 (SEQ ID No. 27)

80 μl of 20 uM DG74 (SEQ ID No. 26)

20 μl of low-DNA Taq polymerase (5 units/μl)

380 μl of ultrafiltered water

The amplification mix is made up as follows (in order):

25 μl 4× PCR mix

25 μl 8 mM MgCl₂

2 drops mineral oil (Sigma #M5904)

50 μl of DNA sample

The amplification conditions in the thermal cycler TC-480 (Perkin Elmer)are:

50° C., 2 minutes (optional)

95° C., 1 minute (optional)

Cycling:

95° C., 1 minute

55° C., 1 minute for 30 to 35 cycles

72° C., 7 minutes to overnight

Detection is performed as described in Example 4.

Although the foregoing invention has been described in some detail forthe purpose of illustration, it will be obvious that changes andmodifications may be practiced within the scope of the appended claimsby those of ordinary skill in the art.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 48                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GTATTCACCGCGGCATGCTGATCCG25                                                   (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 21 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       TATTCACCGCGGCATGCTGAT21                                                       (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       AGTAACCATTTGGAGCTAGCCGT23                                                     (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CGGCTAGCTCCAAAAGGTTACTCTA25                                                   (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CGGCTAGCTCTAAAAGGTTACTCTA25                                                   (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CGACGGCTAGCTCCAAATGGTTACT25                                                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       CACATGCTACAAGGGTCGGTACAGT25                                                   (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       ACTGTACCGACCATTGTAGCATGTG25                                                   (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GGCGCTTACCACTTTGTGATTCATG25                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      GGAGTGGGTTGTACCAGAAGTAGAT25                                                   (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      CTAATCCCATAAAACTATTCTCAGT25                                                   (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      CGGTACAAAGGGCTGCGATGCCG23                                                     (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      GCAAGGAGCCCGCTTACCACGGTAT25                                                   (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      AAGCCGCGAGGCGGAGCCAATCT23                                                     (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      GAGACCGGCTTTCCGAGATTCGCTC25                                                   (2) INFORMATION FOR SEQ ID NO:16:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                      CCAACTTTCATGACTTGACGGG22                                                      (2) INFORMATION FOR SEQ ID NO:17:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                      GGTGTGTACAAGCCCCGGGAACGTA25                                                   (2) INFORMATION FOR SEQ ID NO:18:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                      GCCGGTGGAGTAACCTTTTAGGAGC25                                                   (2) INFORMATION FOR SEQ ID NO:19:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                      CCGGTGGAGTAACCTTTTAGGA22                                                      (2) INFORMATION FOR SEQ ID NO:20:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                      TTGCGACTCGTTGTACCAACCATTG25                                                   (2) INFORMATION FOR SEQ ID NO:21:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                      AACTGAGATTGGCTTTAAGAGATTA25                                                   (2) INFORMATION FOR SEQ ID NO:22:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                      TAATCTCTTAAAGCCAATCTCAGTT25                                                   (2) INFORMATION FOR SEQ ID NO:23:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                      CCGAACTGAGACTGGCTTTAAGAGA25                                                   (2) INFORMATION FOR SEQ ID NO:24:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                      AACTGAGACTGGCTTTAAGAGATTA25                                                   (2) INFORMATION FOR SEQ ID NO:25:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                      AACTGAGACTGGCTTTAAGAGAT23                                                     (2) INFORMATION FOR SEQ ID NO:26:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 19 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                      AGGAGGTGATCCAACCGCA19                                                         (2) INFORMATION FOR SEQ ID NO:27:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 20 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                      AACTGGAGGAAGGTGGGGAT20                                                        (2) INFORMATION FOR SEQ ID NO:28:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 375 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                      GTCATTAGTTGCCATCATTCAGTTGGGCACTCTAATGAGACTGCCGGTGACAAGCCGGAG60                GAAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGACCAGGGCTTCACACGTCATAC120               AATGGTCGGTACAGAGGGTAGCCAAGCCGCGAGGCGGAGCCAATCTCACAAAACCGATCG180               TAGTCCGGATTGCACTCTGCAACTCGAGTGCATGAAGTCGGAATCGCTAGTAATCGCAGG240               TCAGCATACTGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGG300               AGTGGGGGATACCAGAAGTAGGTAGGGTAACCGCAAGGAGCCCGCTTACCACGGTATGCT360               TCATGACTGGGGTGA375                                                            (2) INFORMATION FOR SEQ ID NO:29:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 386 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                      TTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGTGG60                GGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGTTG120               GTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTCGG180               ATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCACG240               CCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTA300               ACACCCGAAGTCGGTGAGGTAACCTTTTAGGAGCCAGCCGCCTAAGGTGGGATAGATGAT360               TGGGGTGACGTCGTAACAAGGTAGCC386                                                 (2) INFORMATION FOR SEQ ID NO:30:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 386 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                      AGTTGCCATCATTTAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGT60                GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGC120               TGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAGTCTCAGTTC180               GGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCA240               CGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTG300               TAACACCCGAAGTCGGTGAGGTAACCGTAAGGAGCCAGCCGCCTAAGGTGGGATAGATGA360               TTGGGGTGAAGTCGTAACAAGGTAGC386                                                 (2) INFORMATION FOR SEQ ID NO:31:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 394 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                      CTTAAGCTTAGTTGCCATCATTAAGTTGGGCACTCTAAGTTGACGCCGGTGACAAACCGG60                AGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGATTTGGGCTACACACGTGCT120               ACAATGGACAATACAAAGGGYAGCGAAACCGCGAGGTCAAGCAAATCCCATAAAGTTGTT180               CTCAGTTCGGATTGTAGTCTGCAACTCGACTATATGAAGCTGGAATCGCTAGTAATCGTA240               GATCAGCATGCTACGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCACG300               AGAGTTTGTAACACCCGAAGCCGGTGGAGTAACCATTTGGAGCTAGCGTCGAAGGTGGGA360               CAAATGATTGGGGTGAGTCGTAACAAGGTAGCCG394                                         (2) INFORMATION FOR SEQ ID NO:32:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                      ACTGAGAATAGTTTTATGGGATTAG25                                                   (2) INFORMATION FOR SEQ ID NO:33:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                      AGGGTAACCTTTATGGAGCCAGCCG25                                                   (2) INFORMATION FOR SEQ ID NO:34:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                      CGGCTGGCTCCATAAAGGTTACCCT25                                                   (2) INFORMATION FOR SEQ ID NO:35:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                      ATCTCTTAAAGCCAATCTCAGTT23                                                     (2) INFORMATION FOR SEQ ID NO:36:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 23 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                      AACTGAGATTGGCTTTAAGAGAT23                                                     (2) INFORMATION FOR SEQ ID NO:37:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 27 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                      GTACAAGGCCCGGGAACGTATTCACCG27                                                 (2) INFORMATION FOR SEQ ID NO:38:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                      TAACCTTTTAGGAGCCAGCCGCCTA25                                                   (2) INFORMATION FOR SEQ ID NO:39:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                      CCTTTTAGGAGCCAGCCGCCTAAGG25                                                   (2) INFORMATION FOR SEQ ID NO:40:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                      CCTTAGGCGGCTGGCTCCTAAAAGG25                                                   (2) INFORMATION FOR SEQ ID NO:41:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                      CGGTTTCGCTGACCCTTTGTATTGT25                                                   (2) INFORMATION FOR SEQ ID NO:42:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 25 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                      AGGGTAACCTTTATGGAGCCAGCCG25                                                   (2) INFORMATION FOR SEQ ID NO:43:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                      GACGTCAAATCATCATGCCCCTTATGTC28                                                (2) INFORMATION FOR SEQ ID NO:44:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                      GACCTAAGGGCCATGATGACTTGACGTC28                                                (2) INFORMATION FOR SEQ ID NO:45:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 28 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                      GACATAAGGGCCATGAGGACTTGACGTC28                                                (2) INFORMATION FOR SEQ ID NO:46:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 22 base pairs                                                     (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                      GGTTAAGTCCCGCAACGAGCGC22                                                      (2) INFORMATION FOR SEQ ID NO:47:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 366 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                      GGGCACTCTAAGTTGACNGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCA60                TCATGCCCCTTATGATTTGGGCTACACACGTGCTACAATGGACAATACAAAGGGCAGCGA120               AACCGCGAGGTCAAGCAAATCCCATAAAGTTGTTCTCAGTTCGGATTGTAGTCTGCAACT180               CGACTACATGAAGCTGGAATCGCTAGTAATCGTAGATCAGCATGCTACGGTGAATACGTT240               CCCGGGTCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGCCGGTG300               GAGTAACCTTTTAGGAGCTAGCNGTCGAAGGTGGGACAAATGATTGGGGTGAGTCGTAAC360               AAGGTA366                                                                     (2) INFORMATION FOR SEQ ID NO:48:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 386 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                      AGTTGCCATCATTAAGTTGGGCACTCTAGCGAGACTGCCGGTAATAAACCGGAGGAAGGT60                GGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGT120               TGGTACAACGAGTCGCAAGCCGGTGACGGCAAGCTAATCTCTTAAAGCCAATCTCAGTTC180               GGATTGTAGGCTGCAACTCGCCTACATGAAGTCGGAATCGCTAGTAATCGCGGATCAGCA240               CGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTG300               TAACACCCGAGTCGGTGAGGTAACCTATTAGGAGCCGCCGCCTAAGGTGGGATAGATGAT360               TGGGGTGAGTCGTAACAAGGTAGCCG386                                                 __________________________________________________________________________

We claim:
 1. A method for determining the presence or absence of abacterium capable of causing meningitis, in a sample suspected ofcontaining said bacterium, said method comprising:(a) amplifying atarget region of a polynucleotide of said bacterium to a detectablelevel using a pair of oligonucleotide primers consisting of thesequences5'-AGGAGGTGATCCAACCGCA-3' (SEQ ID NO: 26),5'-AACTGGAGGAAGGTGGGGGAT-3' (SEQ ID NO: 27), or the complementarysequences thereto; (b) mixing the amplified target region of step (a)with an oligonucleotide probe consisting of the sequences selected fromthe group consisting5'-CTAATCCCATAAAACTATTCTCAGT-3' (SEQ ID NO: 11),5'-AAGCCGCGAGGCGGAGCCAATCT-3' (SEQ ID NO: 14),5'-GGTGTGTACAAGCCCCGGGAACGTA-3' (SEQ ID NO: 17),5'-TTGCGACTCGTTGTACCAACCATTG-3' (SEQ ID NO: 20),5'-TATTCACCGCGGCATGCTGAT-3' (SEQ ID NO: 2),5'-AGTAACCATTTGGAGCTAGCCGT-3' (SEQ ID NO: 3),5'-CGGCTAGCTCCAAAAGGTTACTCTA-3' (SEQ ID NO: 4),5'-CGGCTAGCTCTAAAAGGTTACTCTA-3' (SEQ ID NO: 5),5'-CGACGGCTAGCTCCAAATGGTTACT-3' (SEQ ID NO: 6),5'-CACATGCTACAAGGGTCGGTACAGT-3' (SEQ ID NO: 7),5'-CCAACTTTCATGACTTGACGGG-3' (SEQ ID NO: 16),5'-CCGGTGGAGTAACCTTTTAGGA-3' (SEQ ID NO: 19),5'-AACTGAGATTGGCTTTAAGAGATTA-3' (SEQ ID NO: 21),5'-AACTGAGACTGGCTTTAAGAGATTA-3' (SEQ ID NO: 24),5'-AACTGAGACTGGCTTTAAGAGAT-3' (SEQ ID NO: 25), and the sequencescomplementary thereto (c) incubating the amplified target region withsaid probe under conditions which allow specificity of hybrid duplexessuch that said probe should be capable of detecting bacterial speciesthat cause meningicoccal disease and should be capable of excludingnon-meningicoccal memingicoccal disease-causing bacterial speciescommonly found in cerebrospinal fluid; and (d) detecting hybrids formedbetween said amplified target region and said probe, whereby thepresence or absence of the bacterium is determined.
 2. The method ofclaim 1 wherein the target region is amplified by means of polymerasechain reaction.
 3. An oligonucleotide probe consisting of a sequenceselected from the group consisting of5'-TATTCACCGCGGCATGCTGAT-3' (SEQ IDNO: 2), 5'-AGTAACCATTTGGACTTAGCCGT-3' (SEQ ID NO: 3),5'-CGGCTAGCTCCAAAGGTTACTCTA-3' (SEQ ID NO: 4),5'-CGGCTAGCTCTAAAAGGTTACTCTA-3' (SEQ ID NO: 5),5'-CGACGGCTAGCTCCAAATGGTTACT-3' (SEQ ID NO: 6),5'-CACATGCTACAAGGGTCGGTACAGT-3' (SEQ ID NO: 7),5'-CTAATCCCATAAAACTATTCTCAGT-3' (SEQ ID NO: 11),5'-AAGCCGCGAGGCGGAGCCAATCT-3' (SEQ ID NO: 14),5'-CCAACTTTCATGACTTGACGGG-3' (SEQ ID NO: 16),5'-GGTGTGTACAAGCCCCGGGAACGTA-3' (SEQ ID NO: 17),5'-CCGGTGGAGTAACCTTTTAGGA-3' (SEQ ID NO: 19),5'-TTGCGACTCGTTGTACCAACCATTG-3' (SEQ ID NO: 20),5'-AACTGAGATTGGCTTTAAGAGATTA-3' (SEQ ID NO: 21),5'-AACTGAGACTGGCTTTAAGAGATTA-3' (SEQ ID NO: 24),5'-AACTGAGACTGGCFITAAGAGAT-3' (SEQ ID NO: 25), andthe complementarysequences thereto.
 4. A set of reagents for determining the presence orabsence of a bacterium capable of causing meningitis, in a samplesuspected of containing said bacterium, comprising a pair ofoligonucleotide primers consisting of thesequenccs5'-AGGAGGTGATCCAACCGCA-3' (SEQ ID NO: 26),5'-AACTGGAGGAAGGTGGGGAT-3' (SEQ ID NO: 27), or the complementarysequences thereto; and an oligonucleotide probe selected from the groupconsisting of the sequences5'-TATTCACCGCGGCATGCTGAT-3' (SEQ ID NO: 2),5'-AGTAACCATTTGGACTTAGCCGT-3' (SEQ ID NO: 3),5'-CGGCTAGCTCCAAAGGTTACTCTA-3' (SEQ ID NO: 4),5'-CGGCTAGCTCTAAAAGGTTACTCTA-3' (SEQ ID NO: 5),5'-CGACGGCTAGCTCCAAATGGTTACT-3' (SEQ ID NO: 6),5'-CACATGCTACAAGGGTCGGTACAGT-3' (SEQ ID NO: 7),5'-CTAATCCCATAAAACTATTCTCAGT-3' (SEQ ID NO: 11),5'-AAGCCGCGAGGCGGAGCCAATCT-3' (SEQ ID NO: 14),5'-CCAACTTTCATGACTTGACGGG-3' (SEQ ID NO: 16),5'-GGTGTGTACAAGCCCCGGGAACGTA-3' (SEQ ID NO: 17),5'-CCGGTGGAGTAACCTTTTAGGA-3' (SEQ ID NO: 19),5'-TTGCGACTCGTTGTACCAACCATTG-3' (SEQ ID NO: 20),5'-AACTGAGATTGGCTTTAAGAGATTA-3' (SEQ ID NO: 21),5'-AACTGAGACTGGCTTTAAGAGATTA-3' (SEQ ID NO: 24),5'-AACTGAGACTGGCFITAAGAGAT-3' (SEQ ID NO: 25), and the complementarysequences thereto:such that said probe should be capable of detectingbacterial species that cause meningococcal disease and should be capableof excluding non-memingicoccal disease-causing bacterial commonly foundin cerebrospinal fluid.